The present invention relates to genes which respectively encode the a-subunits of the two isozymes (isoenzymes) of rice anthranilate synthase, as well as to DNAs relating to said genes. Specifically, the present invention relates to two novel DNA segments respectively encoding such proteins which are respectively the xcex1-subunits of the two isozymes, i.e. the first isozyme and the second isozyme, of anthranilate synthase participating in the biosynthesis of tryptophan in rice plants.
Another aspect of the present invention relates to a novel DNA which encodes a novel protein having the activity of the xcex1-subunit protein of the first isozyme of anthranilate synthase. The present invention also relates to a novel recombinant vector in which said novel DNA has been inserted. Further, Escherichia coli, plants and seeds which have been transformed with said novel DNA are embraced within the scope of the present invention.
Another aspect of the present invention relates to a method of increasing the tryptophan content of a plant by the use of the novel DNA of the present invention. Further, the present invention relates to a method of selecting a transformed plant cell containing the novel DNA of the present invention, and to a method of producing a transformed plant containing the novel DNA.
Another aspect of the present invention relates to a DNA which acts as a promoter for the rice anthranilate synthase gene.
Grains such as rice, maize and wheat are important nutrient source for humans and domestic animals. However, their nutritive value is low because they contain only a small amount of tryptophan, which is one of the essential amino acids. Thus, a need exists for a new plant variety capable of producing grain which has a high nutritive value with a high tryptophan content.
In the tryptophan biosynthetic pathway in a plant, anthranilic acid is biosynthesized from chorismic acid. It is known that anthranilate synthase (hereinafter sometimes referred to as ASA) catalyzes the formation of anthranilic acid and tryptophan is formed from anthranilic acid via indole through six-step enzyme reaction [Seikagaku Jikken Koza (Lectures on Experiments in Biochemistry), vol. 11, pp. 652-653 (1976) published by Tokyo Kagaku Dojin].
Plant anthranilate synthase enzymes so far known consist of plural subunits. For example, anthranilate synthase of Arabidopsis (Japanese name: shiroinunazuna, scientific name: Arabidopsis thaliana) is known to consist of two kinds of isozymes, the first isozyme and the second isozyme, each of which is a dimer consisting of the xcex1-subunit and the xcex2-subunit. The gene encoding the xcex1-subunit of the first isozyme of Arabidopsis anthranilate synthase (abbreviated as ASA1) as well as the gene encoding the xcex1-subunit of the second isozyme of Arabidopsis anthranilate synthase (abbreviated as ASA2) (the genes are referred to as asa1 and asa2, respectively) have been isolated, and their DNA sequences have been determined [The Plant Cell, vol. 4, pp. 721-733 (1992)].
On the other hand, we, the present inventors already took an interest in the xcex1-subunit of anthranilate synthase as expected to have a functional domain which plays an important role in the regulation of tryptophan biosynthesis in rice, and we made studies in 1996 to isolate a gene encoding the anthranilate synthase protein, for the purpose of obtaining information about the biosynthetic regulation mechanism of tryptophan and a phytohormone IAA. According to the abstract of the report of these studies, we, the present inventors extracted mRNA and genomic DNA from rice (Norin No.8) as explant, and we prepared a cDNA library and a genomic DNA library, subjected these libraries to the genomic Southern analysis and made screening of the libraries with using cDNA fragments of the Arabidopsis asa gene as the probes, and thereby we obtained DNA which is supposed to be corresponding to the asa gene of rice anthranilate synthase [Ikushu (Breeding), vol. 46, suppl. vol. 2, p. 28 (1996)]. Although it is reported in this abstract that a DNA fragment corresponding to the ASA gene of rice anthranilate synthase was obtained, specific techniques used for obtaining said DNA fragment are not disclosed there, and it is reported that the nucleotide sequence of said DNA fragment has not been determined yet. The abstract of the above report also refers to the presence of two kinds of DNAs which are supposed to be corresponding to the gene for encoding the rice anthranilate synthase [Ikushu (Breeding), vol. 46, suppl. vol. 2, p. 28 (1996)].
There has also been a report on that DNA of the ASA gene encoding the xcex1-subunit of the first isozyme of Arabidopsis ASA and also a DNA fragment as obtained by modifying said DNA are introduced into a tobacco plant with the expression of the function of said gene in tobacco [Massachusetts Institute of Technology, Cambridge, Mass. (1993)].
However, so far as the present inventors are aware of, no report has been made on the analysis of the amino acid sequence of a protein which is the xcex1-subunit of rice ASA isozyme, and on the method which was actually used for obtaining a gene encoding the xcex1-subunit of rice ASA isozyme. The promoter sequence relating to the expression of said gene is not known, either. Further, there has been no report on the utilization of a gene encoding the rice ASA.
One object of the present invention is to obtain from a rice plant a novel gene relating to the rice ASA, specifically, a new DNA for encoding the xcex1-subunit of the first isozyme of the rice ASA. Another object of the present invention is to determine the nucleotide sequence of this DNA.
Another object of the present invention is to provide a novel DNA capable of encoding a novel protein having the activity of the xcex1-subunit of the first isozyme of rice ASA. A further object of the present invention is to transform useful plants such as maize, rice, soybean, wheat, barley, tomato and potato, with said novel DNA, and to provide novel useful transformant plants capable of producing seeds which have a high tryptophan content. A yet further object of the present invention is to construct a novel DNA sequence capable of encoding a protein having the activity of the xcex1-subunit of the first isozyme of rice ASA, and to provide an efficient method for obtaining cells and plants as transformed with said novel DNA.
The other objects of the present invention will be clear from by the descriptions below.
In order to accomplish the above-described objects, the present inventors have made a series of studies. First, a study has been made for obtaining such genes which respectively encode the xcex1-subunits of the two ASA isozymes from rice. In this study, we, the inventors have extracted a total RNA from tissue of an explant of rice such as disrupted green stem and leaf by a known technique for the gene engineering, and we have isolated mRNAs from the extracted total RNAs by a conventional method, and have successfully obtained cDNAs of rice from the mRNAs with using a commercially available cDNA synthesis kit. It has been found through trials and errors that recombinant vectors can be constructed by ligating the above cDNAs into such a phage vector (available from STRATAGENE) as prepared by treating the end of an EcoRI-cleaved fragment of xcex gt11 phage vector with an alkaline phosphatase derived from calf small intestines, and that replicable recombinant xcex phages can be constructed by packaging the obtained recombinant vectors in a xcex phage.
Further, it has been found that a lot of recombinant xcex phages can be obtained in the form of a large number of plaques, by incubation of Escherichia coli Y1088 as infected with the above recombinant xcex phages, and that a group of recombinant xcex phages present in the resultant plaques comprises various phages each containing the rice-derived cDNA and can be utilized as a rice cDNA library.
On the other hand, we, the present inventors have now prepared by chemical synthesis such two oligonucleotides which can be considered to be suitable for use as primers in PCR, and which are namely the first oligonucleotide consisting of 21 nucleotides and the second oligonucleotide consisting of 24 nucleotides, with our reference to the amino acid sequences of the proteins which are respectively the xcex1-subunits of the first and second isozymes of Arabidopsis ASA, as well as the nucleotide sequences of the genes encode said proteins presumable from their amino acid sequences, which are described in the above-mentioned publication [The Plant Cell, vol. 4, pp. 721-733 (1992)].
PCR amplification has been carried out by us with using a mixture of the first and second oligonucleotides mentioned above with a commercially available Arabidopsis cDNA library (utilized as a template). And then it has now been found that the first and second oligonucleotides serve as primers (complementary DNAs) which are necessary in PCR, and that DNA fragments, which constitute parts of the DNA sequences corresponding to the genes encoding the xcex1-subunits of the two isozymes of Arabidopsis ASA, can be amplified by PCR. The resulting products of the amplification of parts of the genes encoding the xcex1-subunits of the first and second isozymes of Arabidopsis ASA have now been successfully recovered from the reaction mixture of PCR, as DNA probes.
The present inventors have succeeded, as a result of many errors and trials, in isolating eight plaques of the recombinant xcex phages carrying the genes respectively encoding the xcex1-subunits of the first and second isozymes of rice ASA, from the previously obtained rice cDNA library (three hundred thousand plaques of recombinant xcex phages mentioned above), by the phage plaque hybridization method with using the DNA probe obtained above. The recombinant xcex phages in said eight plaques have been separately amplified, and then each xcex phage DNA has been isolated by a conventional method.
The DNA fragments are obtained by digesting with the restriction enzyme EcoRI the above recombinant phage DNAs which are carrying the DNA sequences assumable to be corresponding to the genes encoding the xcex1-subunits of the first and second isozymes of rice ASA. Said DNA fragments have been inserted into the EcoRI cleavage site of the commercially available plasmid vector pBluescript II SK(+), by using a DNA ligation kit. Escherichia coli XLI-Blue MRFxe2x80x2 has been transformed with the thus obtained recombinant plasmid vectors, and the resulting transformants have been incubated to give a large number of bacterial cells. Plasmid DNA fragments have been isolated from these cells, and their nucleotide sequences have been analyzed. As a result, we have now confirmed that one of the two DNA sequences, which are contained in the plasmid DNA fragments and assumable to be corresponding to the genes encoding the xcex1-subunits of the first and second isozymes of rice ASA, has the nucleotide sequence shown in SEQ ID NO: 1 of Sequence Listing given hereinafter. It has also now been confirmed that the other one of the above-mentioned two DNA sequences has the nucleotide sequence shown in SEQ ID NO: 10 of Sequence Listing.
Further, so far as the present inventors are aware of, the DNAs having the nucleotide sequences shown in SEQ ID NOS: 1 and 10 of Sequence Listing, respectively, have not been disclosed in any publication, and thus they can be recognized as novel DNA sequences.
The protein, which is encoded by the DNA having the nucleotide sequence shown in SEQ ID NO: 1 of Sequence Listing, is recognized as the protein having the amino acid sequence shown in SEQ ID NO: 2 of Sequence Listing and is also recognized as the protein constituting the xcex1-subunit of the first isozyme of rice ASA.
Accordingly, the first aspect of the present invention provides a DNA encoding a protein which is the xcex1-subunit of the first isozyme of rice anthranilate synthase, and which protein has the amino acid sequence shown in SEQ ID NO: 2 of Sequence Listing.
The DNA according to the first aspect of the present invention can specifically be the DNA having the nucleotide sequence shown in SEQ ID NO: 1 of Sequence Listing.
The new DNA of the first aspect of the present invention is the DNA encoding the protein which is the xcex1-subunit of the first isozyme of rice ASA. This new DNA has now been obtained from the rice cDNA library by recombinant DNA techniques, as described above, based on the study made by the present inventors. However, once the nucleotide sequence of the DNA has now been determined by the present invention, it can also be chemically synthesized from nucleotides, with referring to the nucleotide sequence of SEQ ID NO: 1. It is also possible to produce the DNA of the first aspect of the present invention in a known manner from a rice chromosomal DNA library by the known polymerase chain reaction (PCR) or hybridization, with using as a probe such a synthetic nucleotide as prepared with referring to the nucleotide sequence of SEQ ID NO: 1, or with using the prepared synthetic oligonucleotide as a primer.
The protein which is encoded by the DNA having the nucleotide sequence shown in SEQ ID NO: 10 of Sequence Listing, is recognized as the protein having the amino acid sequence shown in SEQ ID NO: 11 and is also recognised as the protein constituting the xcex1-subunit of the second isozyme of rice ASA.
Accordingly, the second aspect of the present invention provides a DNA encoding a protein which is the xcex1-subunit of the second isozyme of rice anthranilate synthase and which protein has the amino acid sequence shown in SEQ ID NO: 11 of Sequence Listing.
The DNA according to the second aspect of the present invention can specifically be the DNA having the nucleotide sequence shown in SEQ ID NO: 10 of Sequence Listing.
The new DNA of the second aspect of the present invention is the DNA encoding the protein which is the xcex1-subunit of the second isozyme of rice ASA. This new DNA has been obtained from the rice cDNA library by recombinant DNA techniques, as described above, based on the study made by the present inventors. However, once the nucleotide sequence of the DNA has been determined by the present invention, it can also be chemically synthesized from nucleotides, with referring to the nucleotide sequence of SEQ ID NO: 10. It is also possible to produce the DNA of the second aspect of the present invention in a known manner from a rice chromosomal DNA library by the known polymerase chain reaction (PCR) or hybridization, with using as a probe a synthetic nucleotide as prepared with referring to the nucleotide sequence of SEQ ID NO: 10 or with using the prepared synthetic oligonucleotide as a primer.
Next, the process for preparing the DNAs of the first and second aspects of the present invention from stems and leaves of rice plant by the recombinant DNA techniques is outlined below.
(1) Preparation of Rice mRNA and Construction of Rice cDNA Library
Total RNA is extracted from tissues, e.g. stems and leaves, roots and callus, preferably green stems and leaves, of rice plant (Oryza sativa) by a conventional method. After removal of contaminants such as proteins, the total RNA is passed through a column of oligo dT cellulose to purify the poly(A)+RNAs, whereby rice mRNAs can be obtained.
Then, rice cDNAs are synthesized from the above mRNAs with using a commercially available cDNA synthesis kit. The so synthesized cDNAs are ligated into a phage vector such as xcexgt11 vector or xcexZAPII vector, and the resulting recombinant vectors are packaged in a xcex phage. A number of recombinant phages can be thus prepared, and subsegment incubation of Escherichia coli cells as infected with these recombinant phages gives a large number of the recombinant phages as the plaques. The above procedure can be carried out by using a commercially available cDNA cloning kit.
The recombinant phages which are obtained as the plaques of host E. coli cells as described above, comprise various phages containing total rice-derived cDNAs and, therefore, can be used as the rice cDNA library.
(2) Construction of Primers for PCR
We, the present inventors have now constructed by chemical synthesis two kinds of oligonucleotides (the two oligonucleotides shown in SEQ ID NOS: 8 and 9 of Sequence Listing given hereinafter) as primers for PCR (complementary DNAs), while we are referring to the nucleotide sequence which is common to the known nucleotide sequences of the genes encoding the xcex1-subunits of the first and second isozymes of Arabidopsis ASA (on-line data base EMBL: M92353), and while we are taking into account the fact that the first isozyme of Arabidopsis ASA has a higher expression level in a plant.
(3) Preparation of DNA Probes
The DNA probes are then prepared, which are to be used for selectively obtaining the desired DNAs encoding the rice ASA xcex1-subunits from the rice cDNA library previously obtained as said plaques comprising a large number of recombinant phages. For the preparation of said DNA probes, the DNAs constituting some parts of the genes encoding the xcex1-subunits of the first and second isozymes of Arabidopsis ASA are amplified by PCR, with using the above-mentioned synthetic oligonucleotides as the primers and an Arabidopsis cDNA library as the template.
After making repeated amplification reactions by PCR, the products of the amplification of DNA fragments which are some parts of the DNA sequences corresponding to the genes encoding the Arabidopsis ASA xcex1-subunits are recovered from the reaction mixture of PCR as the desired DNA probes.
(4) Selection of cDNA clones of Rice ASA xcex1-Subunit Genes from Rice cDNA Library
The rice cDNA library is thus obtained as a large number of plaques of the recombinant phages in the above, and it is next subjected to a screening by the phage plaque hybridization using the above DNA probes. There can be selected several plaques comprising recombinant phages carrying the DNA sequences which are corresponding, as a whole, to the desired rice ASA xcex1-subunit genes.
The recombinant phages are thus obtained as the selected plaques, and they comprise the DNA fragments carrying the desired DNA sequences which are corresponding to the cDNA colone of the rice ASA xcex1-subunit genes, as explained below.
In more detail, thus, a phage is obtained from each of the plaques selected by the plaque hybridization as described above, and a phage DNA is recovered from said phage. The phage DNA is then treated according to the dideoxy method or the like, to determine the nucleotide sequence of the rice-derived DNA fragment inserted therein. For this, the amino acid sequence determined based on the protein-encoding region (open reading frame) in the nucleotide sequence of the rice-derived DNA insert is compared with the known amino acid sequence of the Arabidopsis ASA xcex1-subunit protein, for the judgment of homology. In this manner, the phage DNAs obtained as above can be specified to be the DNA fragments carrying the DNA sequences which are corresponding to the rice ASA xcex1-subunit genes.
Thus, the DNA insert fragments, which are judged to carry the DNA sequences corresponding to the rice ASA xcex1-subunit genes, can then be obtained by cleavage from the resulting phage DNAs of the phages selected in the above manner, with using restriction enzymes.
(5) Cloning of cDNA Corresponding to Rice ASA xcex1-Subunit Genes
The above DNAs, which have been obtained by cleavage from the phages as the DNA fragments carrying the DNA sequences corresponding to the rice ASA xcex1-subunit genes, are inserted into the EcoRI cleavage site of the plasmid vector pBluescript II SK(+) in order to construct recombinant plasmid vectors. E. coli XL1-Blue MRFxe2x80x2 is transformed with the thus constructed recombinant plasmid vectors. The resulting E. coli transformants are cultured to effect cloning of the above recombinant plasmids comprising the DNA fragments which are carrying the DNA sequences corresponding to the rice ASA xcex1-subunit genes. Thus, the DNA fragments carrying the DNA sequences corresponding to the rice ASA xcex1-subunit genes can be cloned.
According to the present invention, as explained in the above, two kinds of DNA fragments which are different in number of nucleotides were obtained in the form of the DNA fragments, which can be prepared by ligation of them into the plasmid vector pBluescript II SK(+) and cloning of the vector in E. coli as described above, and which DNA fragments carry the DNA sequences corresponding to the rice ASA xcex1-subunit genes. The smaller one of the two DNA fragments thus obtained by us is provisionally named as DNA fragment X, and the larger one is provisionally named as DNA fragment Y.
(6) Sequence Analysis of Cloned DNAs
(i) The DNA fragments X and Y are then separately cleaved from the cloned recombinant plasmids mentioned above with the restriction enzyme EcoRI, as the two kinds of DNA fragments carrying the DNA sequences which are corresponding to the rice ASA xcex1-subunit genes. When the treatment of the DNA fragments X and Y thus cleaved is made by means of a commercially available nucleotide sequence determination kit, the entire sequences of the DNA fragments X and Y carrying the DNA sequences, which are corresponding to the rice ASA xcex1-subunit genes, can be determined. The DNA sequence encoding the xcex1-subunit of the first isozyme of rice ASA was thus determined for the above DNA fragment X, and it exhibits the nucleotide sequence which is shown in SEQ ID NO: 1 of Sequence Listing given below and which consists of 1734 nucleotides. This DNA sequence, which is named as xe2x80x9cOSASA-1 sequencexe2x80x9d, is an example of the DNA according to the first aspect of the present invention.
Incidentally, the above DNA fragment X was obtained in Example 1 given hereinafter, and the DNA fragment X carries the DNA sequence having the nucleotide sequence shown in SEQ ID NO: 1 of Sequence listing and corresponding to the gene encoding the xcex1-subunit of the first isozyme of rice ASA. This DNA fragment X was inserted into the EcoRI cleavage site of the plasmid vector pBluescript II SK(+) (STRATAGENE). The obtained recombinant plasmid vector (named vector pOSASA-1) was introduced into E. coli XL1-Blue MRFxe2x80x2, and the resulting transformant was named Escherichia coli XL1-Blue MRFxe2x80x2, (OS-asa1). This E. coli transformant was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Tsukuba-shi, Ibaraki-ken, Japan on Aug. 18, 1997 with accession number FERM P-16388. Further, Escherichia coli OS-asa1 was deposited with the above depository on Aug. 7, 1998 under the Budapest Treaty with accession number FERM BP-6453.
The DNA according to the first aspect of the present invention is useful in that the use of the information on the nucleotide sequence of the present DNA as determined by the present invention makes it possible to produce of a large amount of the xcex1-subunit protein of the first isozyme of rice ASA by chemical synthesis. And thus the present DNA is capable of contributing to the development of enzymatic studies of the xcex1-subunit protein of the first isozyme of rice ASA.
(ii) The DNA sequence encoding the xcex1-subunit of the second isozyme of rice ASA was decided as described above for the above DNA fragment Y. DNA fragment Y exhibits the nucleotide sequence which is shown in SEQ ID NO: 10 of Sequence Listing and which consists of 1821 nucleotides. This DNA sequence is named xe2x80x9cOSASA-2 sequencexe2x80x9d, and is an example of the DNA according to the second aspect of the present invention.
The above DNA fragment Y was obtained in Example 1, and it carries the DNA sequence having the nucleotide sequence shown in SEQ ID NO: 10 of Sequence Listing and corresponding to the gene encoding the xcex1-subunit of the second isozyme of rice ASA. This DNA fragment Y was inserted into the EcoRI cleavage site of the plasmid vector pBluescript II SK(+). The obtained recombinant plasmid vector(named vector pOSASA-2) as introduced into E. coli XL1-Blue MRFxe2x80x2, and the resulting transformant was named Escherichia coli XL1-Blue MRFxe2x80x2, (OS-asa2). This E. coli transformant was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology on Jun. 18, 1998 with accession number FERM P-16853, and also on Aug. 7, 1998 under the Budapest Treaty with accession number FERM BP-6454.
On the other hand, it is widely recognized in the art that even when a single or plural amino acid residues in the amino acid sequence of a protein having a certain physiological activity are deleted, and/or substituted by other amino acid residues, and/or a single or plural amino acid residues are added to said amino acid sequence, the resulting sequence sometimes retains the original physiological activity of the protein of the original amino acid sequence. Thus, the DNA of the first aspect of the present invention can be a DNA which encodes a protein having the activity of the xcex1-subunit of the first isozyme of rice ASA, even after modification is made to one or several parts of its nucleotide sequence.
In other words, the DNA according to the first aspect of the present invention remains capable of encoding a protein having the activity of the xcex1-subunit of the first isozyme of rice ASA, even after a single or plural nucleotides, for example, 1, 2 or 3 to 10 nucleotides in the nucleotide sequence thereof are altered to other nucleotides.
Accordingly, the third aspect of the present invention provides a DNA encoding a protein having the activity of the xcex1-subunit of the first isozyme of anthranilate synthase and having such an amino acid sequence as formed by modification of the amino acid sequence shown in SEQ ID NO: 2 of Sequence Listing given below, said modification being made by deletion of a single or plural amino acid residues in said amino acid sequence, and/or by substitution of a single or plural amino acid residues in said amino acid sequence by other amino acid residues, and/or by insertion or addition of amino acid residues to said amino acid sequence.
The DNA according to the third aspect of the present invention is a modification of the DNA according to the first aspect of the present invention. It can be obtained by modifying the nucleotide sequence of the DNA of the first aspect of the present invention by a method such as site-specific mutagenesis, so that a modified DNA still will encode a protein having such an amino acid sequence in which amino acid residues at specific positions of the protein as encoded by the modified DNA have been deleted, substituted or added in the above-described manner.
The modified DNA according to the third aspect of the present invention can also be obtained by a method comprising mutating cells containing DNA fragments carrying the DNA of the first aspect of the present invention, and then selecting from the mutated cells such a DNA which can hybridize with the DNA having the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions, and which has a nucleotide sequence partially different from the sequence of SEQ ID NO: 1. The term xe2x80x9cstringent conditionsxe2x80x9d as used herein will refer to the conditions under which so-called specific hybridization with the DNA of the first aspect of the present invention occurs and non-specific hybridization does not occur. Such stringent conditions are difficult to specify numerically, but may include, for example, those conditions which allow such two nucleic acids having a high homology, e.g. such DNAs having 98% or more homology to hybridize with each other, but which do not allow such two nucleic acids having a less homology to hybridize with each other.
Accordingly, an example of the DNA according to the third aspect of the present invention may be such a DNA which has a nucleotide sequence partially different from the nucleotide sequence shown in SEQ ID NO: 1, which has a high homology to the nucleotide sequence of SEQ ID NO: 1, which is capable of hybridizing with the DNA having the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and which encodes a protein having anthranilate synthase activity.
The DNA according to the third aspect of the present invention can be a DNA encoding a protein which has the amino acid sequence shown in SEQ ID NO: 13, and which is such a protein having the activity of the xcex1-subunit of the first isozyme of rice anthranilate synthase but being insensitive to the feedback inhibition by tryptophan.
A specific example of the DNA according to the third aspect of the present invention is the DNA sequence which has the nucleotide sequence of SEQ ID NO: 12 of Sequence Listing, and which was obtained by the method described in Example 2 below and is named as xe2x80x9cmodified D sequencexe2x80x9d therein.
The above DNA, named as the modified D sequence, has the nucleotide sequence of SEQ ID NO: 12 and is the DNA which encodes the above protein having the activity of the xcex1-subunit of the first isozyme of rice ASA but being insensitive to the feedback inhibition by tryptophan.
Also encompassed by the third aspect of the present invention is a DNA fragment encoding such a protein which has been modified in a manner as described above, and which is capable of constituting a holo-enzyme having the anthranilate synthase activity, in association with the anthranilate synthase xcex2-subunit. That is, the third aspect of the present invention may include within its scope a DNA encoding such a protein which has an amino acid sequence as constructed by deletion or substitution or insertion or addition of one to several amino acid residues within the amino acid sequence of SEQ ID NO: 2 and which protein is still capable of constituting a holo-enzyme having anthranilate synthase activity, in association with the anthranilate synthase xcex2-subunit.
When the use of the DNA of the first aspect of the present invention or a part thereof is made as a probe, it is possible that the DNA sequence of the gene of the xcex1-subunit of the first isozyme of ASA is produced from a plant chromosome by a conventional method. The ASA genes as derived from rice chromosome shall comprise the introns as described below. Such a DNA sequence segmented with the introns, which can be obtained in the above manner, is also embraced within the scope of the third aspect of the present invention, so far as the such DNA sequence encodes a protein capable of constituting the holo-enzyme having the anthranilate synthase activity, in association with the anthranilate synthase xcex2-subunit.
By the expression xe2x80x9cASA xcex1-subunit genexe2x80x9d as used herein is meant a DNA encoding the xcex1-subunit protein which is capable of constituting the holoenzyme having anthranilate synthase activity, in association with the rice anthranilate synthase xcex2-subunit. The term xe2x80x9cxcex1-subunitxe2x80x9d as used here will refer to one or both of the xcex1-subunits of the first and second isozymes of rice ASA.
As described above, the DNA of the first aspect of the present invention can be modified into the DNA of the third aspect of the present invention. Likewise, the DNA of the second aspect of the present invention, i.e. the DNA encoding the protein which is the xcex1-subunit of the second isozyme of rice ASA, can also be modified by altering a part of the nucleotide sequence of the DNA of the second aspect invention.
Accordingly, the fourth aspect of the present invention provides a DNA encoding a protein having the activity of the xcex1-subunit of the second isozyme of anthranilate synthase and having such an amino acid sequence as formed by modification of the amino acid sequence shown in SEQ ID NO: 11 of Sequence Listing; said modification being made by deletion of a single or plural amino acid residues in said amino acid sequence, and/or by substitution of a single or plural amino acid residues in said amino acid sequence by other amino acid residues, and/or by insertion or addition of amino acid residues to said amino acid sequence.
The DNA according to the fourth aspect of the present invention can be a DNA which encodes the protein having the activity of the xcex1-subunit of the second isozyme of anthranilate synthase; said DNA having a nucleotide sequence partially different from the nucleotide sequence shown in SEQ ID NO: 10 of Sequence Listing, and said DNA having homology to the nucleotide sequence shown in said SEQ ID NO: 10, and being capable of hybridizing with the DNA having the nucleotide sequence shown in SEQ ID NO: 10, under stringent conditions.
As described above, a specific example of the novel DNA according to the third aspect of the present invention is the DNA sequence which is prepared by the method described in Example 2 and is named as the modified D sequence.
This modified D sequence is the DNA having the nucleotide sequence shown in SEQ ID NO: 12 of Sequence Listing given hereinafter. This modified D sequence corresponds to DNA which is a modified DNA derived from the DNA of the first aspect of the present invention having the nucleotide sequence of SEQ ID NO: 1, in such manner that G (guanine) at nucleotide 967 in the GAC sequence (codon for aspartic acid) at nucleotides 967, 968 and 969 in the sequence of SEQ ID NO: 1 is replaced by A (alanine) so as to provide the AAC a sequence of a codon for asparagine in the positions containing the nucleotide 967 in the sequence of SEQ ID NO:1. The protein encoded by this modified D sequence has the amino acid sequence shown in SEQ ID NO: 13 of Sequence Listing and exhibits the activity of the xcex1-subunit of the first isozyme of rice ASA.
Further, the protein encoded by the modified D sequence according to the third aspect of the present invention is a novel protein whose enzyme activity has been so altered that ASA participating in the tryptophan biosynthetic pathway is made insusceptible of the feedback inhibition by tryptophan which is a biosynthetic product. The DNA encoding this novel protein may be used for the transformation of plants in order to increase the tryptophan content of plants as described after.
Generally, a DNA sequence can be partially altered by known methods such as the Kunkel method (Methods in Enzymology, vol. 154, no. 367) and the oligonucleotide-direct dual amber method.
We, the present inventors have discussed and studied about approaches to the partial alternation of the nucleotide sequence of the DNA of the first aspect of the present invention, for such purpose that the first isozyme of ASA encoded by the DNA of the first aspect of the present invention is modified to be insusceptible of the tryptophan feedback inhibition, while we are referring to the previous report on the known ASA gene of such an Arabidopsis mutant which is resistant to tryptophan analogues [published in xe2x80x9cPlant Physiologyxe2x80x9d, vol. 110, pp. 51-59 (1996)]. As a result, the present inventors have got such an anticipative conception that the modified DNA as prepared by replacing guanine (G) at nucleotide 967 in the nucleotide sequence of SEQ ID NO: 1 (OSASA-1 sequence) by adenine (A) will be effective for the above purpose.
On the basis of this conception, we, the present inventors have made various studies. Through many trials and errors, we have now constructed a recombinant plasmid vector by a method wherein a DNA fragment carrying the DNA sequence which is corresponding to the gene of the xcex1-subunit of the first isozyme of rice ASA, i.e. OSASA-1 sequence, is inserted into the EcoRI cleavage site of the plasmid vector pBluescript II SK(+) by using a ligation kit. This recombinant plasmid vector (hereinafter referred to as pOSASA-1) so obtained has been recognized to be suitable as a starting material for the preparation of the desired novel modified DNA.
For the above purpose and in order to achieve the preparation of the desired novel modified DNA from the above starting material, i.e. the above recombinant plasmid vector pOSASA-1 according to by PCR method, we have prepared the following usable four primers by chemical synthesis; which four primers are primer OSASN1, that is, the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 16 of Sequence Listing given below; and primer OSASN2m, that is, the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 17; and primer OSASC1, that is, the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 18; and primer OSASC2, that is, the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 19 of Sequence Listing.
We, the present inventors have now succeeded in producing the DNA of SEQ ID NO: 12, i.e. the xe2x80x9cmodified D sequencexe2x80x9d, which is an example of the modified DNA of the present invention, when we have carryied out the procedure described in Example 2, with utilizing the above recombinant plasmid vector pOSASA-1 as well as the above-mentioned four kinds of the synthetic oligonucletides as the primers.
Outlined below is the process for modifying DNA which are comprising the steps as described in Example 2 herein after and which can be suitably employed for the preparation of a DNA fragment carrying the above xe2x80x9cmodified D sequencexe2x80x9d, an example of the third aspect of the present invention.
(1) Cloning of the DNA of the First Aspect of the Present Invention
A DNA fragment, which is carrying the DNA of the first aspect of the present invention having the nucleotide sequence of SEQ ID NO: 1 and consisting of the 1734 nucleotides, i.e. the above-mentioned OSASA-1 sequence, is inserted into the EcoRI cleavage site of the vector pBluescript II SK(+) by the use of a DNA ligation kit, thereby to obtain the above-mentioned recombinant plasmid vector pOSASA-1. This vector is introduced into E. coli XLI-Blue MRFxe2x80x2, and the resulting E. coli transformant is cultured. From the cultured cells is isolated a large amount of the plasmid vector pOSASA-1 by means of ordinary extraction. By this procedure, the DNA sequence according to the first aspect of the present invention, i.e. the OSASA-1 sequence can be cloned.
(2) Construction of Primers for RCR
Four kinds of the oligonucleotides having the nucleotide sequences identified below are synthesized as the primers by using a DNA synthesizer (Model-391, Applied Biosystems, Inc.). Thus, the following four primers are prepared by chemical synthesis, which are the primer OSASN1, namely the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 16 of Sequence Listing; the primer OSASN2, namely the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 17; the primer OSASC1, namely the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 18; and the primer OSASC2, namely the oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 19 of Sequence Listing.
(i) Primer OSASAN1 (primer having the following nucleotide sequence of SEQ ID NO: 16): 5xe2x80x2-GAGTCAGTTGACGAAGCGTATGAGG-3xe2x80x2
(ii) Primer OSASAN2 (primer having the following nucleotide sequence of SEQ ID NO: 17): 5xe2x80x2-GTACATTTGCTAACCCCTTTGAGG-3xe2x80x2
(iii) Primer OSASAC1 (primer having the following nucleotide sequence of SEQ ID NO: 18): 5xe2x80x2-CAAAGGGGTTAGCAAATGTACGC-3xe2x80x2
(iv) Primer OSASAC2 (primer having the following nucleotide sequence of SEQ ID NO: 19): 5xe2x80x2-GTTCAACGTTCATCAGTTTCTCCACC-3xe2x80x2
(3) Amplification of the Desired DNA Fragments by PCR and Recovery Thereof
In order to amplify the desired DNA fragments, the following two reactions, namely reactions (A) and (B), are carried out as the first step of PCR.
The reaction (A) is carried out by a method comprising adding the above recombinant plasmid vector pOSASA-1 as the template, as well as the above primer OSASAN1 (the synthetic oligonucleotide of SEQ ID NO: 16) and the above primer OSASAC1 (the synthetic oligonucleotide of SEQ ID NO: 18; wherein GTT at the 8th to 10th positions from the 5xe2x80x2 end of OSASAC1 is capable of inducing the modification part AAC of the xe2x80x9cmodified D sequencexe2x80x9d), to an ordinary reaction mixture for effecting PCR [comprising Tris-HCl, MgCl2, KCl, four kinds of deoxynucleotide triphosphate (dNTP) and La Taq DNA polymerase], and subjecting the resulting mixture to the amplification reaction.
By this reaction (A), a DNA fragment carrying a partial nucleotide sequence of the modified D sequence (said DNA fragment is hereinafter referred to as xe2x80x9cDNA fragment-Axe2x80x9d) may be formed as a product of the amplification.
The reaction (B) is carried out by a method comprising adding the above recombinant plasmid vector pOSASA-1 as the template, as well as the above primer OSASAC2 (the synthetic oligonucleotide of SEQ ID NO: 19) and the above primer OSASAN2 (the synthetic oligonucleotide of SEQ ID NO: 17; wherein AAC at the 12th to 14th positions from the 5xe2x80x2 end of OSASAN2 is capable of inducing the modification part AAC of the modified D sequence), to the same reaction mixture for PCR as that used in the reaction (A), and subjecting the resulting mixture to the amplification reaction.
By this reaction (B), a DNA fragment carrying a partial nucleotide sequence of the modified D sequence (said DNA fragment is hereinafter referred to as xe2x80x9cDNA fragment-Bxe2x80x9d) may be formed as a product of the amplification.
The above amplification reactions by PCR can be carried out by the use of a commercially available PCR apparatus.
After the completion of amplification reactions, the reaction mixture resulting from the reaction (A) is fractionated by low-melting point agarose electrophoresis, and then a band containing the DNA fragment-A of 268 bp (base pairs) as the amplification product is cut out of the agarose gel. The reaction mixture resulting from the reaction (B) is likewise fractionated by low-melting point agarose electrophoresis, and then a band containing the DNA fragment-B of 336 bp (base pairs) is cut out of the agarose gel.
The so obtained two gel pieces are purified by using a DNA purification kit, e.g. Genclean II (Funakoshi), whereby the purified product of the DNA fragment-A and the purified product of the DNA fragment-B, respectively, are obtained.
Further, as the second step of PCR, a reaction is carried out in this step for the purpose of preparing a DNA fragment of 583 bp (base pairs) (fragment C), which is corresponding to a partial sequence of the DNA sequence having such nucleotide sequence wherein guanine is replaced by adenine at the nucleotide 967 of the nucleotide sequence of SEQ ID NO: 1 (thus, said DNA sequence is corresponding to the xe2x80x9cmodified D sequencexe2x80x9d fragment according to the third aspect of the present invention, which has the nucleotide sequence of SEQ ID NO: 12).
This reaction just in the above is carried out by adding, as the templates, the purified product of DNA fragment-A (268 bp sequence) as produced by amplification in the above reaction (A) as well as the purified product of DNA fragment-B (336 bp sequence) as produced by amplification in the above reaction (B), to an ordinary amplification reaction mixture for effecting PCR (comprising Tris-HCl, MgCl2, KCl, four kinds of dNTP and La Taq DNA polymerase), and subjecting the resulting mixture to the amplification reaction. After the completion of reaction, the reaction mixture is fractionated by low-melting point agarose electrophoresis, and then a band containing the desired DNA fragment of 583 bp (hereinafter referred to as xe2x80x9cDNA fragment-Cxe2x80x9d) is cut out of the agarose gel.
The obtained gel piece is purified by using a DNA purification kit, e.g. Genclean II (Funakoshi), thereby to obtain the purified product of the DNA fragment-C. This DNA fragment-C has the nucleotide sequence which is corresponding to a partial sequence of the xe2x80x9cmodified D sequencexe2x80x9d according to the third aspect of the present invention, and which has such structure wherein cleavage sites for the restriction enzymes AflII and BglII are present.
When Cleavage of this DNA fragment-C is made with the restriction enzymes AflII and BglII, there is provided DNA fragment-xcex1 of 288 bp which has the desired nucleotide substitution in its sequence, and which has an AflII cleavage site at the 5xe2x80x2 end, and has a BalII cleavage site at the 3xe2x80x2 end.
(4) Cloning of a DNA Fragment Carrying the Modified D Sequence
The desired DNA fragment carrying the modified D sequence is then prepared by the use of the DNA fragment-C as obtained in the above (3).
First, the DNA fragment-C is treated with the restriction enzymes AflII and BglII, whereby there is isolated a DNA fragment which is carrying a partial sequence of the modified D sequence and which is having an AflII cleavage site at the 5xe2x80x2 end and a BglII cleavage site at the 3xe2x80x2 end. In this way, a DNA fragment sample (i) which contains the DNA sequence corresponding to the modified D sequence as intended is thus obtained.
Then the plasmid vector pOSASA-1 containing the DNA shown in SEQ ID NO: 1 (i.e. OSASA-1 sequence) is treated with the restriction enzymes AflII and BglII, thereby to obtain plasmid fragment (ii) which has a BglII cleavage site at the 5xe2x80x2 end and an AflII cleavage site at the 3xe2x80x2 end, and which comprises a sequence of nucleotides 1 to 933 and a sequence of nucleotides 1220 to 1734 in the DNA sequence of SEQ ID NO: 1.
The so obtained AflII-BglII plasmid fragment (ii) is mixed with the above DNA fragment sample (i) which is containing the DNA sequence corresponding to the modified D sequence. The resulting mixture is subjected to ligation reaction using a DNA ligation kit. Thereby, a recombinant plasmid containing the modified D sequence of SEQ ID NO: 12 (hereinafter referred to as xe2x80x9cplasmid pBluescript-DNA-Dxe2x80x9d) can be constructed.
The so obtained recombinant plasmid, pBluescript-DNA-D, is introduced into E. coli XLI-Blue MRFxe2x80x2. The resulting transformant (hereinafter referred to as Escherichia coli XL1-Blue MRFxe2x80x2/pBluescript-DNA-D) is cultured in a liquid medium to give a large number of transformant cells. The E. coli cells thus proliferated are containing copies of the above recombinant plasmid pBluescript-DNA-D. The modified D sequence can be cloned in this manner. A plasmid containing the modified D sequence is isolated from the cultured E. coli cells by means of ordinary extraction.
(5) Recovery of a DNA Fragment Carrying the Modified D Sequence
The plasmid containing the modified D sequence as obtained in the above (4) is then digested with the restriction enzyme EcoRI.
This treatment gives a reaction mixture containing a DNA fragment which is carrying the modified D sequence and which is having the nucleotide sequence ATG at the 5xe2x80x2 end adjacent to the EcoRI cleavage site and also having an extended part containing an EcoRI cleavage site at the 3xe2x80x2 end.
The above reaction mixture is fractionated by low-melting point agarose electrophoresis, and a band containing the above DNA fragment is cut out of the agarose gel. The obtained agarose gel piece is dissolved in TE buffer. The resulting solution is extracted with phenol, whereby the above DNA fragment is recovered as an extract. The phenol extract containing the above DNA fragment is mixed with a 3 M aqueous solution of sodium acetate and ethanol. The resulting mixture is allowed to stand at 20xc2x0 C. about 6 hours and then centrifuged at a low temperature, whereby the above DNA fragment is precipitated. By drying the precipitate, the desired DNA fragment which carries the modified D sequence, is obtained as powder. This powdery DNA fragment carrying the modified D sequence is soluble in water.
Described above is a process for preparing the DNA fragment carrying the modified D sequencer which is an example of the DNA of the third aspect of the present invention, by utilizing the recombinant DNA techniques. However, the desired DNA can also be prepared by a known method for the chemical synthesis of polynucleotides, with referring to the nucleotide sequence shown in SEQ ID NO: 12 of Sequence Listing.
The foregoing are explanations of the mode for carrying out the third aspect of the present invention, in respect of such case when the modification is made to effect the xe2x80x9cguanine to adenine changexe2x80x9d at the nucleotide 967 in the sequence of the DNA of the first aspect of the present invention shown in SEQ ID NO: 1. It is also possible to prepare another modified DNA which contains a nucleotide substitution at a position different from the nucleotide 967 in the sequence of the DNA of the first aspect of the present invention shown in SEQ ID NO: 1, if the use of the DNA shown in SEQ ID NO: 1 is made as the template and then a combination of several synthetic oligonucleotides having appropriately designed nucleotide sequences is used as the primers.
We, the present inventors have made further studies. As a result, we have now found that both of the novel DNA of the first aspect of the present invention which encodes the xcex1-subunit of the first isozyme of rice ASA, as well as the novel modified DNA of the third aspect of the present invention which is derived from the DNA encoding the xcex1-subunit of the first isozyme of rice ASA can be introduced into a plant, when the novel DNA of this invention is incorporated in a recombinant vector, and also that the novel DNA can be expressed in the plant. To this end, there may be utilized the known techniques in biotechnology for the transformation of a plant, which comprise introduction of an exogenous gene and expression of the exogenous gene in the resultant transgenic plant.
Accordingly, the fifth aspect of the present invention provides a transformed plant, characterized by having a plant cell as transformed by introduction of such a recombinant vector which carries the DNA of the first aspect of the present invention for encoding the xcex1-subunit of the first isozyme of rice anthranilate synthase; said DNA as introduced being capable of expression therein.
The sixth aspect of the present invention provides a transformed plant, characterized by having a plant cell as transformed by introduction of such a recombinant vector which carries carrying the DNA of the third aspect of the present invention for encoding a protein having the activity of the xcex1-subunit of the first isozyme of rice anthranilate synthase but being insensitive to the feedback inhibition by tryptophan, and particularly, such a recombinant vector which carries the DNA for encoding the protein having the amino acid sequence shown in SEQ ID NO: 13 or the DNA having the nucleotide sequence shown in SEQ ID NO: 12 and, said DNA as introduced can be expressed therein.
Further, it has been found that when the transformed plant according to the fifth or sixth aspect of the present invention is a plant capable of producing seeds by culturing, seeds of said transformed plant can be harvested by culturing said plant under ordinary conditions.
Accordingly, the seventh aspect of the present invention provides seeds of a transformed plant which are harvested from culturing of transformed plant which is produced by introducing such a recombinant vector carrying the DNA of the first aspect of the present invention for encoding the xcex1-subunit of the first isozyme of rice anthranilate synthase, or such a recombinant vector carrying the modified DNA of the third aspect of the present invention, into a plant cell, and in which transformed plant said DNA can be expressed.
The eighth aspect of the present invention provides a recombinant vector which comprises an inserted DNA fragment carrying the DNA sequence having the nucleotide sequence shown in SEQ ID NO: 1 or 10 of Sequence Listing, and which vector is capable of expressing said DNA sequence in a host cell.
The ninth aspect of the present invention provides a recombinant vector which comprises an inserted DNA fragment carrying such DNA sequence as named the modified D sequence having the nucleotide sequence shown in SEQ ID NO: 12 of Sequence Listing, and which vector is capable of expressing said DNA sequence in a host cell.
The tenth aspect of the present invention provides, as a novel microorganism, E. coli as transformed with such a recombinant vector which comprises an inserted DNA fragment carrying the DNA sequence having the nucleotide sequence shown in SEQ ID NO: 1 or 10 of Sequence Listing, and which vector is capable of expressing said DNA sequence in a host cell.
The eleventh aspect of the present invention provides, as a novel microorganism, E. coli as transformed with such a recombinant vector which comprises an inserted DNA fragment carrying the DNA sequence as named the modified D sequence having the nucleotide sequence shown in SEQ ID NO: 12 of Sequence Listing, and which vector is capable of expressing said DNA sequence in a host cell.
By proliferating the E. coli transformant according to the tenth aspect of the present invention, or the E. coli according to the eleventh aspect of the present invention, a large number of clones of the recombinant vector contained in the cells can be harvested. Examples of the E. coli according to the tenth aspect of the present invention include the above-mentioned Escherichia coli XL1-Blue MRFxe2x80x2 (OS-asa-1) and Escherichia coli XL1-Blue MRFxe2x80x2 (OS-asa-2), which have been deposited under the Budapest Treaty with accession numbers FERM BP-6453 and FERM BP-6454, respectively.
An example of the E. coli transformant according to the eleventh aspect of the present invention is the above-mentioned Escherichia coli XL1-Blue MRFxe2x80x2/pBluescript-DNA-D, which has been deposited under the Budapest Treaty with accession number FERM BP-6451.
The DNA according to the first aspect of the present invention and the DNA according to the third aspect of the present invention can be used as the exogenous genes for making the transformation of a wide variety of plants. Introduction of the DNAs of the present invention as the exogenous genes into plants for the transformation can be carried out by known techniques in biotechnology.
Outlined below is the process described in Example 3 which can be suitably employed for the introduction of the DNA according to the first or third aspect of the present invention, as an exogenous gene into rice plants.
(a) Construction of Recombinant Vectors for Introduction of Exogenous Genes
A known plasmid vector pUBA [Plant Molecular Biology, vol. 18, no. 4, pp. 675-689 (1992)], which contains the known maize ubiquitin promoter, 1st intron and NOS terminator as well as a phosphinothricin resistance gene and an ampicillin resistance gene capable of expressing its effect only in microorganisms, is treated with the restriction enzymes BamHI and SacI in a buffer. This treatment gives such a vector fragment of about 4.8 kb which has been cleaved at the BamHI cleavage site located downstream of the ubiquitin promoter and downstream of the 1st intron and also cleaved at the SacI cleavage site located upstream of the NOS terminator.
An aqueous solution of the so obtained vector DNA fragment is mixed with an aqueous solution of a DNA fragment carrying the DNA of the present invention. The resultant mixture is subjected to ligation reaction by using a DNA ligation kit. This reaction results in the construction of a recombinant vector containing the DNA fragment carrying the DNA of the present invention which is inserted between the ubiquitin promoter and the NOS terminator region of the vector DNA fragment.
The thus constructed recombinant vector is introduced into E. coli JM109, to obtain an E. coli transformant.
The obtained E. coli transformant is inoculated into a medium containing the antibiotic ampicillin and cultured, whereby several ampicillin-resistant E. coli colonies are obtained. These colonies are separately proliferated in a medium containing ampicillin.
Plasmids are isolated from the proliferated ampicillin-resistant E. coli cells of the respective colonies. The plasmids thus recovered include various plasmids, wherein the DNA is inserted in different orientations. The plasmids as recovered from the respective colonies are digested with appropriate restriction enzymes. The resulting reaction mixtures containing various DNA fragments resulting from the digestion are subjected to agarose gel electrophoresis. By analysis of the size and nucleotide sequence of these DNA fragments, there can be selected appropriate plasmids (about 6.5 kb) wherein the DNA of the present invention is inserted downstream of the ubiquitin promoter of the recombinant plasmid in the normal orientation.
The plasmid, wherein the DNA of the first aspect of the present invention shown in SEQ ID NO: 1 is incorporated, is named as vector pUBdW1; and the plasmid, wherein the DNA of the second aspect of the present invention shown in SEQ ID NO: 10 is incorporated is named as vector pUBdW2; and the plasmid, wherein the DNA of the modified D sequence shown in SEQ ID NO: 12 is incorporated, is named as vector pUBdD.
Further, for effecting the preparation of a recombinant vector for Use in the gene introduction according to the Agrobacterium method; a known plasmid vector pIG121-Hm [Plant Cell Physiol., vol. 31, pp. 805-813 (1990)] containing a hygromycin resistance gene is treated with the restriction enzymes PmeI and SacI in a buffer, thereby to obtain a vector fragment of about 9.8 kb.
Each of the above plasmid vectors pUBdW1, pUBdW2 and pUBdD is treated with SphI and SacI in a buffer, followed by the treatment for blunting the SphI-cleaved end, and there is afforded a vector fragment in which the DNA of the present invention is ligated downstream of the ubiquitin promoter and the 1st intron.
Ligation reaction, production of E. coli transformants and recovery of plasmids are carried out in the same manner as described above, with using the above vector fragments carrying the DNA of the present invention. Thereby, their can be obtained the recombinant vectors for the gene introduction according to the Agrobacterium method in which the DNA of the present invention is inserted in the normal orientation.
The plasmid, wherein the DNA of the first aspect of the present invention shown in SEQ ID NO: 1 is incorporated, is named as vector pUb-OSASAW1; and the plasmid, wherein the DNA of the second aspect of the present invention shown in SEQ ID NO: 10 is incorporated, is named as vector pUb-OSASAW2; and the plasmid, wherein the DNA of the modified D sequence of the third aspect of the present invention shown in SEQ ID NO: 12, is incorporated, is named as vector pUb-OSASA1D.
(b) Preparation of Rice Callus
After mature seeds of rice are hulled, the resulting rice seeds with coats are sterilized with an ethanol solution and then with a dilute aqueous solution of sodium hypochlorite, followed by washing with sterilized water.
The rice seeds with coats are placed on such a callus formation medium as prepared by adding sucrose, 2,4-PA as a phytohormone and agar to MS medium. Cultivation of seeds is carried out at 28xc2x0 C. for 40-50 days with irradiation with sunlight at 1500-2500 lx for 15-18 hours per day, affording callus. The callus thus formed are cut from the albumen of the seeds.
(c) Introduction of an Exogenous Gene into Rice Callus Cells
In order to introduce the recombinant vector carrying the normally inserted DNA of the present invention which has been prepared by the method described in the above (a) (i.e. the above-mentioned vector pUb-OSASAW1, pUb-OSASAW2 or pUb-OSASA1D) into callus cells according to the known Agrobacterium method, the recombinant vector is first introduced into Agrobacterium tumefaciens as a host according to the known electroporation technique [Shokubutsu Soshiki Baiyo (Plant Tissue Culture), vol. 10, no. 2, pp. 194-196 (1993)].
The DNA of the present invention can be introduced into rice callus cells by co-cultivation of the thus obtained Agrobacterium with the callus cells as obtained in the above (b), according to a known method [Saibo Kogaku (Cell Engineering), suppl. vol. xe2x80x9cProtocol for Experiments Using Model Plantsxe2x80x9d, pp. 93-98 (1996) published by Shujunsha]. Hygromycin-resistant plant cells as transformed with the DNA of the present invention are thus obtained.
(d) Reselection of Transformed Plant Cells
From the hygromycin-resistant transformed plant cells obtained as above are reselected such transformed plant cells containing a sufficiently effective amount of the DNA of the present invention as the exogenous gene.
To this end, the transformed cells obtained as above are transplanted to a reselecting medium which has been prepared by adding sucrose, 2,4-PA, Gel lite and a tryptophan analogue 5MT (5-methyltryptophan) to N6 medium.
The transplanted cells are cultured there at 25-28xc2x0 C. for 25-30 days with irradiation with light at 2000 lx for 16 hours per day.
The transformed plant cells, which contain a sufficiently effective amount of the DNA of the present invention as the exogenous gene, are resistant to 5MT, and they can grow on a medium containing 5MT which acts as a cell growth inhibitor. The cultured plant cells, which are resistant to 5MT grown on the above 5MT-containing medium, are selected in this way.
(e) Plant Regeneration from 5MT-Resistant Transformed Plant Cells Reselected
The 5MT-resistant cultured plant cells as reselected in the above manner are then transplanted to a differentiation medium for plant regeneration which has been prepared by adding sucrose, benzyladenine as a phytohormone, naphthaleneacetic acid and Gerite to MS medium for plant tissue culture.
The transplanted cells are then cultured at 25-28xc2x0 C. for 25-30 days with irradiation with light at 2000 lx for 16 hours per day, whereby buds and roots can be regenerated from the cultured transformant plant cells by differentiation.
Plumules containing the regenerated buds and roots have grown to a length of 10-30 mm, and thereafter the plumules are transplanted to a habituation medium which has been prepared by adding sucrose and Gerite to MS medium. Cultivation is carried out at 25-28xc2x0 C. for 18-20 days with irradiation with light at 2000 lx for 16 hours per day.
Transformed plants can be regenerated in this manner. The thus obtained transformed plants normally grow when they are transplanted into the soil in a greenhouse and are cultured under ordinary conditions. They can produce rice seeds after 3-6 months of cultivation.
(f) Confirmation of the Introduced Exogenous Gene
Green leaves are taken from the transformed rice plants as regenerated in the above manner The leaves are frozen in liquid nitrogen, followed by disruption. DNA is extracted from the disrupted leaves according to the method of J. Sambrook, et al. [Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press (1989)].
Separately, an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 14 of Sequence Listing, and an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 15, which are to be used as primers, are chemically synthesized.
PCR is carried out in the usual way with using the DNA extracted from the regenerated rice plants as above, as the template, and using the above two synthetic oligonucleotides as the primers in order to amplify the above DNA. The resulting amplification reaction mixture is fractionated by agarose electrophoresis in a conventional manner. A band containing a DNA fragment corresponding to the DNA of the introduced exogenous gene, among various DNA fractions derived from the DNA extracted from the regenerated rice plants, is cut out of the agarose gel.
It is possible to confirm whether the DNA fragment contained in the resultant band corresponds to the DNA of the present invention or not, by analyzing its nucleotide sequence by the known Southern analysis technique.
Extraction of tryptophan from plants and determination of tryptophan content of plants can be carried out by a known method [the Hopkins-Cole method, Seikagaku Jikken Koza (Lectures on Experiments in Biochemistry), vol. 11, published by Tokyo Kagaku Dojin], or by a method utilizing HPLC as described in the example given below. The procedures for the extraction of tryptophan and the determination of tryptophan content can be appropriately modified according to the kind, part and growth stage of a test plant.
The processes for the selection of cells as transformed by introduction of the DNA of the present invention and for the production of the transformed plants are described below.
(a) Construction of a Recombinant Vector for Selection
A recombinant vector, which is to be used for directly introducing a gene into a plant cell for the production of a transformed plant, is constructed in the following manner. That is, a known plasmid vector pBI221 (Clontech), which contains the known cauliflower mosaic virus 35S promoter and NOS terminator, as well as an ampicillin resistance gene cabafle of expressing its effect only in microorganisms, is treated with the restriction enzymes XbaI and SacI in a buffer. This treatment gives a vector fragment of about 3.8 kb which has been cleaved at the XbaI cleavage site located downstream of the 35S promoter and cleaved at the SacI cleavage site located upstream of the NOS terminator.
An aqueous solution of the obtained vector DNA fragment is mixed with an aqueous solution of a DNA fragment carrying the DNA of the third aspect of the present invention (the modified D sequence). The resulting mixture is subjected to ligation reaction by using a DNA ligation kit. This reaction results in the construction of a recombinant vector containing the DNA fragment which carries the DNA of the present invention (the modified D sequence) inserted between the 35S promoter and the NOS terminator region of the vector DNA fragment.
The thus constructed recombinant vector is introduced into E. coli JM109, to obtain an E. coli transformant.
The obtained E. coli transformant is inoculated into a medium containing the antibiotic ampicillin and is cultured, whereby several ampicillin-resistant E. coli colonies are obtained. These colonies are separately proliferated in a medium containing ampicillin.
Plasmids are isolated from the proliferated ampicillin-resistant E. coli cells of the respective colonies. The plasmids thus recovered include various plasmids wherein the DNA is inserted in different orientations. The plasmids as recovered from the respective colonies are digested with appropriate restriction enzymes. The resulting reaction mixtures containing various DNA fragments resulting from the digestion are subjected to agarose gel electrophoresis. By analysis of the size and nucleotide sequence of these DNA fragments, an appropriate plasmid (about 5.6 kb), wherein the DNA of the present invention (the modified D sequence) is inserted downstream of the 35S promoter of the recombinant plasmid in the normal orientation, can be selected. For the recombinant vector for selection and use in the production of a transformed plant, the above vector pUBdD can be employed in the case of the direct gene introduction into plant cells. The above vector pUb-OSASA1D can be employed in the case of the Agrobacterium method.
(b) Preparation of Rice Calluses
After mature seeds of rice are hulled, the resulting rice seeds with coats are sterilized with an ethanol solution and then with a dilute aqueous solution of sodium hypochlorite, followed by washing with sterilized water.
The rice seeds with coats are placed on a callus formation medium which has been prepared by adding sucrose, 2,4-PA as a phytohormone and agar to MS medium. Cultivation is carried out at 28xc2x0 C. for 40-50 days with irradiation with sunlight at 1500-2500 lx for 15-18 hours per day, to afford callus. The callus thus formed are cut from the albumen of the seeds.
(c) Introduction of Recombinant Vectors usable for Selection into Rice Callus Cells
The recombinant vector usable for the selection and carrying the normally inserted DNA of the present invention, which is prepared by the method described in the above (a), i.e. the vector pUBdD, is introduced into the callus cells by the known method for direct introduction with whiskers (Japanese Published Unexamined Patent Application No. . . . /98). The selecting recombinant vector pUb-OSASA1D is introduced into callus cells by the known Agrobacterium method [Saibo Kogaku (Cell Engineering), suppl. vol. xe2x80x9cProtocol for Experiments Using Model Plantsxe2x80x9d, pp. 93-98 (1996) published by Shujunsha].
(d) Selection of Transformed Plant Cells
The callus cells which are containing the recombinant vector usable for selection and which are obtained as above, are added onto and evenly spread over a selecting medium prepared by adding sucrose, 2,4-PA, Gerite and a tryptophan analogue as a selective drug in an amount of 10 mg/1-200 mg/l, preferably 30 mg/1-50 mg/l, to N6 medium. Cultivation is carried out at 25-28xc2x0 C. for 20-60 days, preferably 25-30 days, in a dark place or with irradiation with light at 2000 lx for 16 hours per day.
Plant cells as transformed with the recombinant vector usable for selection, which are resistant to the tryptophan analogue, are thus selected.
(e) Selection of Transformed Plants
In order to obtain the target transformed plants from the resulting tryptophan-analogue-resistant and transformed plant cells obtained as above, these transformed plant cells are transplanted to such a selective differentiation medium for plant regeneration which has been prepared by adding sucrose, benzyladenine as a phytohormone, naphthaleneacetic acid, Gerite and a tryptophan analogue as a selective drug in an amount of 10 mg/1-200 mg/l, preferably 30 mg/1-50 mg/l, to MS medium.
The transplanted plant cells are cultured at 25-28xc2x0 C. for 25-30 days with irradiation with light at 2000 lx for 16 hours per day. Thereby, buds and roots can be regenerated from the cultured transformant plant cells, by differentiation.
Plumules containing the regenerated buds and roots have grown to a length of 10-30 mm, and then the plumules are transplanted to a habituation medium as prepared by adding sucrose and Gerite to MS medium. Cultivation is carried out at 25-28xc2x0 C. for 18-20 days with irradiation with light at 2000 lx for 16 hours per day.
Transformed plants as intended can be regenerated in this manner.
The DNA according to the present invention can be introduced as an exogenous gene, not only into the above-mentioned rice plants but also into other kinds of plants for the transformation of them.
Further, the use of the DNA according to the present invention for the increase of tryptophan content and for the selection of appropriate rice plant is not limited to the above-mentioned rice plants, but it can be extended to other kinds of plants.
Given below are general descriptions of the process for introducing the DNA of the present invention into general plants, and of the process for increasing the tryptophan content, as well as of the process for selecting transformed cells and transformed plants.
There is no specific limitation to the kind of plants into which the DNA of the present invention can be introduced. Representative plants include monocotyledons such as rice, maize, wheat and barley, and dicotyledons such as tobacco, soybean, cotton, tomato, Chinese cabbage, cucumber and lettuce. It is convenient to first prepare the cultured cells from these plants and then introduce the DNA of the present invention as an exogenous gene into the cultured cells.
The cultured cells to be used for the introduction of the DNA of the present invention can be prepared from any explant derived from a plant. For example, such explants derived from scutellum, meristem, pollen, anther, lamina, stem, petiole and root can be used.
It is convenient to introduce the DNA of the present invention into the cultured cells obtained by cultivation of said explant on a callus formation medium, for example, a medium which is prepared by adding a phytohormone such as 2,4-PA (2,4-dichlorophenoxyacetic acid) in an amount of 0.1-5 mg/l, a carbon source such as sucrose in an amount of 10-60 g/l and Gerite in an amount of 1-5 g/l, to a medium for plant tissue culture containing inorganic salts and vitamins as essential components, e.g. MS medium [Murashige, et al. xe2x80x9cPhysiologia Plantarumxe2x80x9d (1962), vol. 15, pp. 473-497], R2 medium [Ojima, et al. xe2x80x9cPlant and Cell Physiologyxe2x80x9d (1973), vol. 14, pp. 1113-1121] or N6 medium [Chu, et al. (1978) xe2x80x9cIn Proc. Symp. Plant Tissue Culture, Science Press Pekingxe2x80x9d, pp. 43-50].
Preferred plant cells, which are usable for the introduction of the DNA of the present invention, include dedifferentiated cultured cells such as callus and suspended cells, cultured cells such as adventitious embryo and shoot primordium, as well as callus cells and suspended cells prepared from cells of plant tissues such as leaf, root, stem, embryo and meristem.
In the process for preparing the cultured cells for the introduction of the DNA of the present invention by cultivation of an explant on a callus formation medium, there is no specific limitation to the cultivation time. However, in view of the necessity of regenerating a transformed plant, it is required that the plant regeneration from said cultured cells be permissible, namely, that the cultured cells be obtained within the period during which the plant cells can retain the capability for plant regeneration.
The cultured cells for the introduction of the DNA of the present invention can be the suspended cells as cultured in a liquid medium, so far as they are the cultured cells having retained the capability for plant regeneration.
In order to introduce the DNA of the present invention into a plant cell, it is necessary first to construct a recombinant vector by inserting the DNA of the present invention into an expression vector. The recombinant vector to be used here needs to have such a structure that the DNA of the present invention is located downstream of an expression promoter and a terminator is located downstream of said DNA, so that the DNA of the present invention can be expressed in a plant after being introduced therein. Useful recombinant vectors include various vectors which are employed for ordinary plant transformation according to the kind of methods for the introduction of DNA into plants. For example, plasmid vectors replicable in E. coli, such as pUC plasmids and pBR322 plasmids, are preferably used in the direct DNA introduction by the electroporation technique or the techniques utilizing particle gun or whisker. And, plasmid vectors such as plan plasmids are preferably used in the DNA introduction by the Agrobacterium method.
Examples of the promoters, which is to be located upstream of the DNA of the present invention in the recombinant vector, include CaMV35S derived from cauliflower mosaic virus [The EMBO Journal, vol. 16, pp. 3901-3907 (1987); Japanese Published Unexamined Patent Application No. 315381/94]; maize ubiquitin promoter (Japanese Published Unexamined Patent Application No. 79983/90); and phaseolin promoter [Plant Cell, vol. 1, pp. 839-853 (1989)]. Suitable terminator, which is to be located downstream of the DNA of the present invention, includes the terminator derived from cauliflower mosaic virus; and the terminator derived from a nopaline synthase gene [The EMBO J., vol. 6, pp. 3901-3907 (1987)]. Nevertheless, these can be used any of the promoters and terminators which function in plants.
In order to efficiently select such plant cells as transformed by the introduction of the DNA of the present invention, it is preferred to introduce into the plant cells the above recombinant vector together with a plasmid vector carrying an appropriate selective marker gen. Useful examples of the selective marker genes include a hygromycin phosphotransferase gene which is resistant to the antibiotic hygromycin; a neomycin phosphotransferase gene which is resistant to kanamycin and gentamicin; and an acetyltransferase gene which is resistant to the herbicide phosphinothricin [The EMBO Journal, vol. 6, pp. 2513-2518 (1987); Japanese Published Unexamined Patent Application No. 171188/90].
Representative methods for the introduction of the DNA of the present invention as an exogenous gene into plant cells are the Agrobacterium method [Bio/technology, vol. 6, pp. 915-922 (1988)]; electroporation [Plant Cell Rep., vol. 10, pp. 106-110 (1991)]; the particle gun method [Theor. Appl. Genet., vol. 79, pp. 337-341 (1990)]; and the whisker method. However, the DNA introduction techniques are not restricted to these methods.
In order to efficiently reselect the transformed cells containing a sufficiently effective amount of the recombinant vector carrying the DNA of the present invention, cultivation is carried out on a medium containing a tryptophan analogue which is a cell growth inhibitor.
An example of the tryptophan analogue is 5-methyltryptophan (5MT), which can be added to the reselecting medium to a concentration of 10 mg/1-1000 mg/l, preferably 20 mg/1-100 mg/l.
Plants are regenerated from the thus reselected transformed plant cells which are containing the recombinant vector carrying the DNA of the present invention inserted as the exogenous gene, as well as the vector carrying the selective marker. The plant regeneration can be carried out by a known method, for example, by cultivation of the transformed plant cells as reselected above on a known medium for the plant regeneration.
The transformed cells are placed on the medium for the plant regeneration and are cultured at 15-30xc2x0 C., preferably 20-28xc2x0 C., for 20-60 days, preferably 30-40 days, with irradiation with light at 500-2000 lx, preferably 800-1000 lx.
In this manner, a plant, which has been transformed by the introduction of the recombinant vector carrying the exogenous gene comprising the DNA of the present invention, can be regenerated from each plant cell.
The plants as regenerated from the transformed cells are then cultured on a habituation medium. After the habituation, the regenerated plants are grown in a greenhouse under ordinary conditions. By 3-6 months of cultivation in the greenhouse, the regenerated plants grow into maturation and become capable of producing seeds.
The presence of the introduced exogenous gene in the thus regenerated and cultured transformant plant can be confirmed by analyzing the nucleotide sequence of the DNA present in the plant, according to the known PCR and Southern analysis techniques [Southern, xe2x80x9cJ. Mol. Biol.xe2x80x9d, vol. 98, pp. 503-517 (1975)].
In the above analysis process, extraction of the DNA from the transformant plant can be carried out by the known method of J. Sambrook, et al. [Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press (1989)].
Upon effecting the analysis of the exogenous gene comprising the DNA of the present invention which is present in the regenerated plant, according to PCR, the DNA which has been extracted from the regenerated plant as described above is used as the template, and synthetic oligonucleotides having nucleotide sequences which are appropriately selected based on the nucleotide sequence of the DNA or the modified DNA of the present invention, are used as the primers. A mixture of said template and primers is added to a reaction mixture effecting for PCR and is subjected to the amplification reaction. In the amplification reaction procedure, DNA denaturation, annealing and extension reaction are repeated several tens of times, whereby the product of amplification of the DNA fragment carrying the DNA sequence of the present invention can be obtained.
The reaction mixture of PCR containing the amplification product is fractionated by means such as agarose electrophoresis to afford fractions of various DNA fragments amplified. A band containing such a DNA fragment, which is recognized to carry the DNA sequence corresponding to the DNA of the present invention a the introduced exogenous gene, is cut out of the agarose gel. By analyzing the nucleotide sequence of the DNA sequence in the DNA fragment contained in the obtained agarose gel piece according to the Southern analysis, it can be confirmed whether or not said DNA sequence corresponds to the DNA of the present invention.
Tryptophan analogues are useful as the selective drug for the selection of the transformed cells and for the selection of the transformed plants according to the present invention. Examples of such useful drugs include the tryptophan analogues as well as their biosynthetic intermediates such as 5-methyltryptophan (5MT), 4-methyltryptophan (4MT), 6-methyltryptophan (6MT), 7-methyltryptophan (7MT), 6-methylanthranilic acid (6MA), 5-methylanthranilic acid (5MA), 3-methylanthranilic acid (3MA), 5-fluoroanthranilic acid (5FA) and 6-fluoroanthranilic acid (6FA).
As described above, the novel DNA according to the third aspect of the present invention can be introduced as an exogenous gene into a plant or plant cell in order to cause the transformation of plant or plant cell which will give a plant or plant cell having an increased tryptophan content.
Accordingly, the twelfth aspect of the present invention provides a method of increasing the tryptophan content of a plant, which comprises: introducing a recombinant vector into a plant cell callus, such a recombinant vector wherein said recombinant vector carries the DNA of the third aspect of the present invention for encoding a protein which is insensitive to the feedback inhibition by tryptophan and which protein has the activity of the xcex1-subunit of the first isozyme of anthranilate synthase, and particularly, the DNA encoding the protein having the amino acid sequence of SEQ ID NO: 13 or the DNA having the nucleotide sequence of SEQ ID NO: 12 of Sequence Listing and wherein said recombinant vector is capable of expressing said DNA in a plant; and thus obtaining a plant callus cell as transformed with said DNA; and regenerating a plant from said plant cell.
The thirteenth aspect of the present invention provides a method of selecting a transformed plant cell, which comprises: introducing a recombinant vector into plant cells to confer or said plant cells the resistance to such a tryptophan analogue that can inhibit the growth of plant cells, wherein said recombinant vector carries the DNA of the third aspect of the present invention for encoding the protein which is insensitive to the feedback inhibition by tryptophan and which protein has the activity of the xcex1-subunit of the first isozyme of anthranilate synthase, and wherein said recombinant vector carries particularly the DNA which encodes the protein having the amino acid sequence of SEQ ID NO: 13 or the DNA having the nucleotide sequence of SEQ ID NO: 12 of Sequence Listing, and wherein said recombinant vector further carries an antibiotic resistance gene and said recombinant vector is capable of expressing said DNA in a plant; and then selecting such transformed which express the resistance to said tryptophan analogue.
The fourteenth aspect of the present invention provides a method of producing a transformed plant having an increased tryptophan content, which comprises: introducing a recombinant vector into plant cells to confer on said plant cells the resistance to the tryptophan analogue that can inhibit the growth of plant cells, wherein said recombinant vector carries the DNA of the third aspect of the present invention for encoding the a protein which is insensitive to the feedback inhibition by tryptophan and which protein has the activity of the xcex1-subunit of the first isozyme of anthranilate synthase, and wherein said recombinant vector carries particularly the DNA for encoding the protein having the amino acid sequence of SEQ ID NO: 13 or the DNA having the nucleotide sequence of SEQ ID NO: 12 of Sequence Listing, and wherein said recombinant vector further carries an antibiotic resistance gene and said recombinant vector is capable of expressing said DNA in a plant; selecting such transformed cells which express the resistance to said tryptophan analogue; and regenerating plants from the thus selected transformed cells.
We, the present inventors have made further studies with the purpose of isolating such a promoter DNA which is useful for the expression of the gene encoding the xcex1-subunit of the first isozyme of rice ASA. As a result, we have now succeeded in obtaining a DNA fragment carrying a promoter DNA sequence which is effective for the expression of the gene encoding the xcex1-subunit of the first isozyme of rice ASA, according to the following procedure. Outlined below are the steps for obtaining said DNA fragment. (A detailed description of the procedure is given in Example 5 hereinafter.)
(a) Preparation of Rice Genomic DNA
Genomic DNA is extracted from tissues, e.g. stems and leaves, roots and calli, preferably stems and leaves or callus, of rice (Oryza sativa) by a conventional method. After removal of contaminants such as proteins, the genomic DNA is further purified by ultracentrifugation.
(b) Preparation of Rice Genomic DNA Fragments
The purified genomic DNA obtained as above is partially digested with the restriction enzyme EcoRI, and the digestion product is subjected to agarose gel electrophoresis. The thus fractionated DNA fragments are transferred to a nylon membrane High Bond N, followed by denaturation to fix the DNA fractions on the membrane.
Each DNA fraction as fixed on the membrane is then subjected to the hybridization reaction with the DIG-labeled probe DNA as prepared from Arabidopsis as in Example 1(5) below. Thereby a DNA fragment emitting the signal can be detected at a DNA size of about 6 kb on the membrane. Such DNA fraction in the agarose gel which is corresponding to this DNA fraction emitting the signal, is partially digested with the restriction enzyme RcoRI in the agarose gel and then cut out of the gel. The thus obtained DNA is purified, and the resulting purified product of the DNA fragment a is dissolved in TE buffer, to obtain fractionated genomic DNA.
(c) Construction of Rice Fractionated Genomic DNA Library
The so obtained genomic DNA fractions are ligated into a phage vector. The resulting recombinant vectors are packaged in a xcex phage. Incubation of E. coli cells as infected with the resultant recombinant xcex phages is then made to give a large number of the recombinant xcex phages, which can be utilized as a fractionated genomic DNA library of rice.
(d) Selection of a Promoter Gene from the Rice Genomic DNA Library
A recombinant phage carrying a DNA sequence corresponding to the promoter gene for the rice ASA gene can be obtained when the above recombinant phages as constructed as the rice genomic DNA library is subjected to screening by the plaque hybridization with utilizing the DIG-labeled probe DNA which has been prepared from Arabidopsis in Example 1(5). As a result of such screening, we have fortunately succeeded in harvesting three phage plaques presumably carrying the promoter gene for the ASA gene, from one hundred thousand phage plaques of said rice genomic DNA library.
These three plaques as harvested are separately digested with the restriction enzyme RcoRI, to give reaction mixtures which are respectively containing EcoRI-digested DNA fragments.
(e) Cloning of Genomic DNA
The EcoRI-digested DNA fragments obtained as above are then ligated into the EcoRI cleavage site of the plasmid vector pBluescript II SK(+). The resulting recombinant plasmid vectors are introduced into E. coli for cloning, followed by isolation of the recombinant plasmid vector clones from E. coli. 
(f) Sequence Analysis of Cloned Plasmid DNA
The recombinant plasmid vector clones obtained as above are then digested with the restriction enzymes RcoRI and BamHI, followed by effecting the nucleotide sequence analysis of the resulting EcoRI-BamHI fragments.
From the genomic DNA clones obtained above could be isolated such a DNA fragment carrying the DNA sequence of the promoter region which acts for the expression of the gene encoding the xcex1-subunit of the first isozyme of rice ASA (said DNA fragment is provisionally referred to as xe2x80x9cDNA fragment Zxe2x80x9d), with reference to its nucleotide sequence.
The entire nucleotide sequence of the thus obtained xe2x80x9cDNA fragment Zxe2x80x9d carrying the promoter DNA was determined by means of an ordinary sequencing kit. The so determined sequence was recognized to be the nucleotide sequence shown in SEQ ID NO: 3 of Sequence Listing The promoter DNA carried by this DNA fragments was recognized to have the nucleotide sequence shown in SEQ ID NO: 7 of Sequence Listing, by referring to the known nucleotide sequence of the DNA of the promoter region of the gene which encodes the xcex1-subunit of the first isozyme of Arabidopsis.
It was confirmed by the test of Example 5 that the entire or partial nucleotide sequence of the xe2x80x9cDNA fragment Zxe2x80x9d had a promoter activity.
The DNA fragment Z carrying the promoter DNA having the nucleotide sequence of SEQ ID NO: 7 was cloned in pBluescript II SK(+) plasmid vector. The resulting recombinant vector was introduced into E. coli XLI-Blue MRFxe2x80x2. The obtained E. coli transformant was named Escherichia coli (Os-asa#7), and it was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology on Aug. 18, 1997 with accession number FERM P-16387 and also on Aug. 7, 1998 under the Budapest Treaty with accession number FERM BP-6452.
(g) Promoter Activity Test
The promoter activity test is now carried out for such promoter region of the gene encoding the xcex1-subunit of the first isozyme of ASA, which is carried by the above DNA fragment Z as isolated from the said rice genomic DNA clone in the above, in order to confirm that it can function as a promoter. Thus, the above DNA fragment Z was inserted into a restriction enzyme cleavage site of the commercially available pBI101 plasmid vector (Clontech) carrying xcex2-glucuronidase gene, which is a reporter gene. There was constructed a recombinant plasmid vector. This recombinant plasmid vector was introduced into plant cells, e.g. rice cultured cells, by a conventional method. It can be confirmed that said promoter region is effective to achieve the expression of the GUS activity, when using a commercially available GUS activity determination kit.
The fifteenth aspect of the present invention provides a DNA which has the nucleotide sequence shown in SEQ ID NO: 7 of Sequence Listing and has a promoter activity for the expression of the rice anthranilate synthase gene.
The sixteenth aspect of the present invention provides a DNA which has the nucleotide sequence shown in SEQ ID NO: 3 of Sequence Listing and comprises a DNA region having a promoter activity for the rice anthranilate synthase gene, as well as the exon DNA sequences and the intron DNA fragment for said DNA region having the promoter activity.
It is widely recognized in the art that even when one to several nucleotides in the nucleotide sequence of DNA having the promoter activity are deleted, substituted, inserted or added, the resulting modified sequence sometimes retains the promoter activity.
The DNAs according to the fifteenth and sixteenth aspects of the present invention can include such DNA fragments which result from such modifications and express the promoter activity in plant cells and plants. Namely, the fifteenth aspect of the present invention includes within the scope such modified DNA having the promoter activity and having a nucleotide sequence as formed by modification of the nucleotide sequence shown in SEQ ID NO: 7, wherein said modification is made by deletion, substitution, insertion or addition of one to several nucleotides. Such modified DNA can be obtained when the nucleotide sequence of the promoter DNA of the present invention is modified by a method such as site-specific mutagenesis, so that amino acid residues at specific positions will be deleted, substituted or added. The promoter activity test can be carried out for the modified DNA in the same manner as above.
In accomplishing the present invention, the ASA isozyme xcex1-subunit genes of the present invention and the promoter sequence for them were isolated from the cDNA library or the genomic DNA library, as described above. As the nucleotide sequence of the promoter DNA has been determined by the present invention as shoun in Sequence Listing, the promoter DNA can also be prepared by chemical synthesis, with reference to the nucleotide sequence shown in Sequence Listing. It is also possible to obtain the promoter DNA from a rice cDNA library or a rice chromosomal DNA library by the known PCR when using a synthetic oligonucleotide primer as prepared based on the above-mentioned nucleotide sequence.